Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print May 19, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.028035
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/3/1009    most recent
biolreprod.104.028035v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Seligman, J.
Right arrow Articles by Kosower, N. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seligman, J.
Right arrow Articles by Kosower, N. S.
Agricola
Right arrow Articles by Seligman, J.
Right arrow Articles by Kosower, N. S.
BIOLOGY OF REPRODUCTION 71, 1009–1015 (2004)
DOI: 10.1095/biolreprod.104.028035
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Tyrosine Phosphorylation, Thiol Status, and Protein Tyrosine Phosphatase in Rat Epididymal Spermatozoa

Judith Seligman, Yehudit Zipser, and Nechama S. Kosower1

Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel-Aviv University, Tel Aviv 69978, Israel


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sperm thiol oxidation and the ability to undergo protein tyrosine phosphorylation are associated with the acquisition of sperm motility and fertilizing ability during passage of spermatozoa through the epididymis. Phosphotyrosine levels in various cells are controlled by tyrosine kinase versus phosphatase, with the latter known to be inhibited by oxidation. In the present paper we examine whether changes in thiol status during sperm maturation affect rat sperm protein phosphotyrosine levels and protein phosphotyrosine phosphatase (PTP) activity. Tyrosine phosphorylation, as demonstrated by immunoblotting (IB), was significantly increased in several sperm tail proteins during maturation in the epididymis. Sperm thiol oxidation with diamide enhanced tail protein phosphorylation; reduction of disulfides with dithiothreitol diminished phosphorylation. In the sperm head, a moderate increase in tyrosine phosphorylation was accompanied by altered localization of phosphotyrosine proteins during maturation. Blocking of thiols and PTP activity with N-ethylmaleimide led to increased tyrosine phosphorylation of protamine in caput sperm heads. Several PTP bands were identified by IB. In the caput spermatozoa, a prominent level of the 50 kDa band was present, whereas in the cauda spermatozoa a very low level of the 50 kDa band was found. PTP activity, measured by using p-nitrophenyl phosphate as a substrate, was significantly higher in the caput spermatozoa (high thiol content) than in the cauda spermatozoa (low thiol content). Our results show that PTP activity is correlated with sperm thiol status and suggest that tyrosine phosphorylation of sperm proteins during sperm maturation is promoted by thiol oxidation and diminished PTP.

epididymis, gamete biology, phosphatases, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphorylation of protein tyrosine residues plays an essential role in the regulation of diverse cellular functions. In spermatozoa, phosphorylation of protein tyrosine residues is associated with sperm capacitation and acrosome reaction in several mammalian species [1–4]. During epididymal maturation, spermatozoa acquire the ability to become motile, and upon capacitation they undergo a qualitative change in motility and gain fertilizing ability [5]. It is thought that phosphorylation/dephosphorylation events, mainly in flagellar proteins, play a role in these motility changes [6, 7].

Two opposing enzyme families, phosphotyrosine kinases (PTKs) and phosphotyrosine phosphatases (PTPs) control the level of any particular protein phosphotyrosine [8–10]. Normally, the activity of cellular PTP is very high relative to that of PTK, so that a very low basal level of phosphotyrosine is maintained in cells. Tyrosine phosphorylation in cells is promoted by binding of ligands to their cellular receptors. A variety of oxidative stress agents such as hydrogen peroxide [11, 12], the thiol oxidizing agent diamide [13, 14], thiol alkylating agents [14, 15], and nitric oxide [16, 17], as well as UV irradiation [18], can also promote protein tyrosine phosphorylation in various intact cells.

Spermatozoa, like other cells, produce reactive oxygen species when incubated under aerobic conditions [15]. A causal association between the formation of reactive oxygen species, tyrosine phosphorylation, and sperm function was found [11]. In general, oxidizing conditions enhanced tyrosine phosphorylation and promoted capacitation and egg penetration, whereas reducing conditions had the opposite effect [11].

Several studies have suggested that stabilization of sperm structures during epididymal maturation is achieved mainly through the oxidation of thiol groups. We have previously demonstrated that in the rat, sperm protein thiols are oxidized upon passage from caput to the cauda epididymis [19, 20]. The potential of spermatozoa to undergo tyrosine phosphorylation is also increased upon passage from caput to cauda epididymis [21–23].

In some cell types, the enhanced tyrosine phosphorylation was suggested to result from oxidation and direct activation of PTK [24, 25]. However, since all PTPs contain at their active center a highly reactive cysteine residue that is essential for phosphatase activity [10, 26], inhibitory effect of the oxidants on PTP activity has been considered to be the more likely mechanism, indirectly responsible for the tyrosine phosphorylation [14, 26, 27]. Many protein kinases such as cAMP-dependent and cAMP-independent protein kinases, casein kinase II, protein kinase C, and PTK have been identified in the spermatozoa of a variety of mammals [21, 28–30]. Relatively little is known about protein phosphatases in mammalian spermatozoa.

In erythrocytes, it has been demonstrated that alterations in cellular thiol status affect the cell phosphotyrosine status and that oxidative stress-induced tyrosine phosphorylation involves inhibition of PTP [14]. In the present work we studied rat sperm tyrosine phosphorylation and PTP during sperm maturation in the epididymis (i.e., in caput and cauda spermatozoa containing high and low thiol levels, respectively) and modulation by altered thiol status. We show that thiol oxidation/reduction affects sperm tyrosine phosphorylation and that PTP activity is correlated with sperm thiol status. Our results suggest that protein tyrosine phosphorylation during sperm maturation is promoted by thiol oxidation and loss of PTP activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Spermatozoa

Mature albino Wistar-derived rat males 4–8 mo old were used for this study. The study was carried out in accordance with the rules established by the Animal Care and Use Committee at Tel-Aviv University. Spermatozoa from caput and cauda epididymis were obtained by cutting the epididymal duct. Spermatozoa were released into 135 mM NaCl, 10 mM NaPO4 buffer, pH 7.4 (PBS), containing 5 mM glucose (PBS-G), washed twice in PBS-G by centrifugation at 600 x g for 5 min, and resuspended in PBS-G. Sperm samples were examined under a microscope to assess cell purity and were counted to assure an equal number of cells in each sample. For most experiments, spermatozoa were obtained from a single rat male, and each experiment was repeated at least three times using a different male for each experiment. For protamine extractions, pooled spermatozoa from two males were used for each experiment; these experiments were repeated three times.

Sperm Protein Phosphotyrosine and PTP Analysis by Immunoblotting

Aliquots of equal number of sperm (2–6 x 106 sperm/ml) were lysed in 5% SDS and 3% of 2-mercaptoethanol (ME). Other aliquots were incubated for 30 min with and without 1 mM of the disulfide reducing reagent dithiothreitol (DTT) or 1 mM of the thiol oxidizing reagent diamide, then lysed. Sperm lysates were sonicated for 10 sec, and then equal volumes were analyzed on SDS-PAGE as described previously [20].

Analysis of phosphotyrosine and PTP was carried out on nitrocellulose membranes following gel transfer. Nitrocellulose membranes were blocked for 1 h at room temperature in a solution of 50 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween-20 (T-TBS) containing 1% milk, then incubated overnight with monoclonal antiphosphotyrosine antibody (BioMakor, Rehovot, Israel) or with monoclonal anti-PTP-1B antibody (FG6-1G, Oncogene Science, Cambridge, MA) as previously described [14, 31]. After washing with blocking solution, the membranes were incubated for 1 h with goat anti-mouse peroxidase-conjugated antibodies (Amersham Biosciences, Little Chalfont, England), washed in T-TBS, and analyzed using the ECL detection system (Amersham). In some experiments, membranes were stripped off and reprobed with monoclonal anti-{alpha} tubulin antibody (Sigma, Rehovot, Israel).

Isolation of Sperm Heads and Extraction of Protamine

Sperm protamine was extracted from sperm heads as previously described [32]. Briefly, for the isolation of sperm heads, spermatozoa were incubated with and without 10 mM of the thiol alkylating reagent N-ethylmaleimide (NEM) for 40 min at 37°C, washed and resuspended in Tris buffer (10 mM Tris-HCl/150 mM NaCl, pH 7.4) with 10 mM DTT. Spermatozoa were sonicated (to separate heads from tails), incubated for 30 min at room temperature in the presence of 1% mixed alkyl-trimethylammonium bromide, and washed. The pellets, which contained sperm heads, were lysed in 6 M guanidinium hydrochloride (GnHCl), 0.3 M of ME, and 0.5 M Tris-HCl, pH 8.9. To extract protamine, 0.25 M HCl was added to sperm head lysates. Samples were incubated overnight, and HCl-soluble proteins were precipitated with 20% trichloroacetic acid, recovered by centrifugation, and washed with acidified acetone and acetone. Protamine was analyzed by electrophoresis on acid-urea PAGE as described previously [32, 33].

Determination of PTP Activity

PTP activity was determined by using p-nitrophenyl phosphate (p-NPP) as a substrate according to published procedures [34]. Sperm suspensions in PBS-G were incubated for 30 min in the presence and absence of 10 mM NEM. Spermatozoa were then washed and suspended in 25 mM Hepes buffer, pH 7.3, 1.0 mM DTT, and 0.1 mM PMSF, and reaction was started by the addition of 15 mM p-NPP to the samples. Samples were incubated at 37°C for 30 min, and the reaction was terminated by the addition of 0.1 M NaOH. The samples were centrifuged (9000 x g), and the release of p-nitrophenol from p-NPP was measured spectroscopically as absorbance (optical density [O.D.]) at 410 nm.

Immunofluorescence Detection of Sperm Head Phosphotyrosine

Caput and cauda sperm suspensions were fixed in 1% paraformaldehyde for 20 min at room temperature and incubated for 10 min with 0.05% NP-40 and PBS containing 1% BSA (PBS-A). Spermatozoa were washed in PBS-A and incubated for 2 h with antimonoclonal phosphotyrosine antibodies at a concentration of 1:50 in PBS-A. Spermatozoa were washed with PBS and incubated for 15 min with anti-mouse IgG antibodies conjugated with fluorescein isothiocyanate at a concentration of 1/500. Spermatozoa were then washed twice with PBS, mounted on a microscope slide, and analyzed using an Olympus fluorescence microscope (Olympus, Tokyo, Japan).

Sperm Fractionation with Triton X-100

Sperm membranes were extracted with Triton X-100 according to published methods [35, 36], with some modifications. Sperm isolated from caput and cauda epididymis were suspended in cold 25 mM Hepes buffer, pH 7.3, containing 1 mM DTT, 1 mM PMSF, and 0.5% Triton X-100. Sperm suspensions were sonicated for 10 sec, incubated on ice for 30 min, and centrifuged at 15 000 x g. The detergent insoluble pellets were washed in Hepes buffer and resuspended in the original volume. Pellet suspensions and supernatant fractions were dissolved in 5% SDS and 3% ME and analyzed by SDS-PAGE, followed by immunoblotting for the identification of PTP.

Densitometric Analysis

Quantification of the protein bands was obtained by densitometry of the scanned Coomassie blue-stained gels and of the scanned immunoblot films. Level of the phosphotyrosine in any of the analyzed bands was expressed as relative to the Coomassie blue-stained protein band. In some experiments, the level of phosphotyrosine was expressed as relative to {alpha}-tubulin level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tyrosine Phosphorylation of Caput and Cauda Sperm Tail Proteins

Spermatozoa from caput and cauda were released from the epididymal duct into PBS, washed, and resuspended in PBS-G. Aliquots of sperm suspensions were lysed immediately; other aliquots were incubated for 30 min at 37°C before lysis. Sperm phosphotyrosine proteins were detected by immunoblotting using antiphosphotyrosine antibodies. Immunoblotting demonstrated several tyrosine phosphorylated protein bands of about 90, 37–27, and 20 kDa in caput and cauda spermatozoa. These bands exhibited higher levels of tyrosine phosphorylation in the cauda spermatozoa, as compared with the caput spermatozoa, in both incubated and nonincubated spermatozoa (Fig. 1). Densitometric analysis (phosphotyrosine levels relative to {alpha}-tubulin levels) showed no significant difference between incubated and nonincubated samples. Significant differences were observed between cauda and caput spermatozoa; the levels of phosphotyrosine in the cauda sperm were 150% ± 12%, 160% ± 16%, and 855% ± 65% of those in the caput for the 90, 37–27, and 20 kDa, respectively (mean ± SEM; n = 3).



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 1. Phosphotyrosine in rat caput and cauda sperm proteins. Caput and cauda spermatozoa were lysed before (0 min) or after incubation for 30 min at 37°C (30 min). Sperm lysates were run on 12% polyacrylamide gel electrophoresis and Western blots were reacted with phosphotyrosine antibody, then stripped off and reacted with {alpha}-tubulin antibody. The results are representative of three independent experiments

Thiols in caput spermatozoa can be oxidized by diamide, and cauda sperm disulfides reduced by DTT [15, 19, 37]. To examine the effect of thiol oxidation and disulfide reduction on sperm phosphotyrosine proteins, spermatozoa were incubated in the presence and absence of diamide or DTT and lysed (see Materials and Methods). Following electrophoresis on SDS-PAGE, protein bands were identified by Coomassie blue staining. Multiple, similar protein bands, ranging from about 100 to 15 kDa, were identified in caput and cauda spermatozoa (Fig. 2A). These proteins were mainly sperm tail proteins [20]. The Coomassie blue-stained protein profiles of spermatozoa incubated with diamide or DTT were similar to the profiles of control spermatozoa incubated in the absence of the reagents (Fig. 2A). Following treatment of caput spermatozoa with diamide, the level of tyrosine phosphorylation was increased (Fig. 2B, lanes 1 and 2). Following reduction of cauda sperm protein disulfides by DTT, the level of tyrosine phosphorylation was decreased (Fig. 2B, lanes 3 and 4). The levels of phosphotyrosine in the diamide-treated caput sperm were on average 150% of those in the control spermatozoa; reduction of disulfides in the cauda spermatozoa led to diminution of tyrosine phosphorylation to about 60% of the levels in the control cells for the 90 and 37–27 kDa protein bands and completely inhibited the phosphorylation of the 20 kDa band (as estimated by densitometry of phosphotyrosine levels relative to Coomassie blue staining).



View larger version (87K):
[in this window]
[in a new window]
  		FIG. 2. Phosphotyrosine in rat caput and cauda sperm proteins following oxidation of thiols and reduction of disulfides. A) Coomassie blue stain; (B) Phosphotyrosine immunoblot. Caput spermatozoa incubated without and with diamide (lanes 1 and 2, respectively); cauda spermatozoa incubated without and with DTT (lanes 3 and 4, respectively). Sperm lysates were run on 15% polyacrylamide gel electrophoresis, and Western blots were reacted with phosphotyrosine antibodies. The results are representative of three independent experiments

Tyrosine Phosphorylation in Protamine Extracted from Caput and Cauda Sperm Heads

Sperm protamine, the major sperm basic head protein, was isolated from caput and cauda sperm heads following treatment of spermatozoa with DTT, as described in Materials and Methods. As can be seen in Figure 3A, tyrosine phosphorylation in protamine extracted from cauda sperm was similar to that in caput sperm protamine (no significant differences were observed by densitometry). Because a high concentration of DTT is used for the isolation of heads prior to protamine extraction (see Materials and Methods), the differences in the level of protamine thiols between caput and cauda spermatozoa [19] were abolished. Under these conditions, PTP would be activated to a similar extent in caput and cauda sperm heads, diminishing any possible native, inherent differences.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 3. Phosphotyrosine in caput and cauda sperm protamine. Urea-PAGE of protamine extracted from caput and cauda rat sperm heads. A) Without NEM treatment; (B) with prior thiol blocking with NEM. Left, Coomassie blue stain; right, phosphotyrosine immunoblot. The results are representative of three independent experiments

To further probe the relation of protamine thiol/disulfide levels to protamine tyrosine phosphorylation and to the activity of PTP, we blocked thiols and PTP activity with NEM prior to isolation of sperm heads and protamine extraction. As can be seen in Figure 3B, following treatment of spermatozoa with NEM, protamine extracted from caput sperm demonstrated a higher phosphotyrosine level than that of protamine extracted from cauda epididymis (phosphotyrosine level in caput sperm protamine was 192% ± 10.1% of the level in the cauda; n = 3). The higher level of tyrosine phosphorylation in the caput than in the cauda sperm protamine observed when PTP was not active indicates that PTP is normally more active in the caput sperm head than in the cauda sperm head. These results are consistent with the notion that the lower PTP activity in cauda sperm allows increased protamine tyrosine phosphorylation.

Immunofluorescence Localization of Phosphotyrosine Proteins in Caput and Cauda Sperm Heads

Immunofluorescence localization of phosphotyrosine proteins in caput and cauda sperm heads is demonstrated in Figure 4. The level of phosphotyrosine proteins in the cauda sperm heads was somewhat higher than in caput sperm heads. The localization of phosphotyrosine proteins in caput sperm heads was quite different from that in the cauda sperm heads. In the caput spermatozoa, phosphotyrosine proteins were localized to the tip of the sperm head and in the connecting piece. In mature cauda spermatozoa, phosphotyrosine proteins were mostly localized to the posterior region of the head and in the connecting piece. No differences in the localization of phosphotyrosine proteins in caput and cauda sperm tails were observed (data not shown).



View larger version (64K):
[in this window]
[in a new window]
  		FIG. 4. Micrographs of phosphotyrosine in caput and cauda sperm heads. Rat spermatozoa were fixed and incubated with phosphotyrosine antibodies, washed, and reacted with anti-mouse IgG conjugated with fluorescein (see Materials and Methods). Sperm fluorescence was examined by fluorescence microscopy and photographed under similar excitation and exposure time. The results are representative of three independent experiments

PTP Protein and Activity in Spermatozoa Isolated from the Caput and Cauda Epididymis

To examine whether changes in tyrosine phosphorylation during maturation are related to changes in PTP, we studied PTP expression in caput and cauda spermatozoa by immunoblotting and measured its activity. To characterize PTP protein in caput and cauda sperm, we analyzed PTP in whole spermatozoa and in sperm heads using anti-PTP-1B antibody. Three major PTP protein bands of about 50, 40, and 27 kDa were observed in caput spermatozoa (Fig. 5, lane 1), whereas in cauda spermatozoa one major band of about 27 kDa was observed (Fig. 5, lane 2). In the caput and cauda sperm head fractions, two major bands of about 27 and 20–18 kDa were observed (Fig. 5, lanes 3 and 4).



View larger version (81K):
[in this window]
[in a new window]
  		FIG. 5. PTP in rat caput and cauda sperm. Spermatozoa were lysed immediately following isolation and analyzed on 12% polyacrylamide SDS-PAGE. A) Coomassie blue stain; (B) PTP-1B immunoblot. Whole sperm isolated from caput and cauda spermatozoa (lanes 1 and 2, respectively); sperm heads isolated from caput and cauda spermatozoa (lanes 3 and 4, respectively). The results are representative of four independent experiments

To examine whether thiol oxidation/reduction can affect the PTP protein profile, spermatozoa were incubated with and without DTT or diamide and analyzed by immunoblotting. As can be seen in Figure 6, no significant effect was observed following thiol oxidation of caput spermatozoa with diamide (Fig. 6, lane 2 as compared with lane 1) and by reduction of cauda sperm protein thiols by DTT (Fig. 6, lane 4 as compared with lane 3).



View larger version (77K):
[in this window]
[in a new window]
  		FIG. 6. Thiol oxidation/reduction and sperm PTP-1B profile. A) Coomassie blue stain; (B) PTP-1B immunoblot. Caput spermatozoa incubated without and with diamide (lanes 1 and 2, respectively); cauda spermatozoa incubated without and with DTT (lanes 3 and 4, respectively). Sperm lysates were run on 10% polyacrylamide gel electrophoresis, and Western blots were reacted with PTP-1B antibodies. The results are representative of three independent experiments

In order to characterize the differences between the caput and cauda sperm PTP protein, we carried out sperm fractionation using Triton X-100. Under the extraction conditions used, the supernatant fraction contains mostly sperm plasma membranes and soluble cytoplasmic components, whereas the pellet fraction contains sperm internal structures and organelles. As can be seen in Figure 7, following Triton X-100 extraction the major PTP bands of about 27 kDa were observed in the pellets in both caput and cauda spermatozoa. The 50 kDa PTP protein band was observed in supernatant prepared from caput spermatozoa, whereas no 50 kDa band was observed in cauda spermatozoa. Thus, our results suggest that the 50 kDa PTP fraction is associated with sperm plasma membranes, whereas the PTP fractions of lower molecular masses are associated with internal sperm structures.



View larger version (74K):
[in this window]
[in a new window]
  		FIG. 7. PTP-1B in rat caput and cauda sperm fractions. Caput and cauda spermatozoa were incubated in lysis buffer containing Triton X-100 and centrifuged (see Materials and Methods). Fractions were run on 12% SDS-PAGE, transferred to nitrocellulose membrane, and blotted with PTP-1B antibodies. Left, pellets (pellet); right, supernatants (supernat). The results are representative of three independent experiments

PTP activity in caput and cauda spermatozoa was measured using p-nitrophenyl phosphate (p-NPP) as a substrate. PTP activity was observed in both caput and cauda spermatozoa. The gross PTP activity in caput spermatozoa was two-fold higher than that of cauda sperm PTP activity (0.93 ± 0.09 and 0.41 ± 0.16 O.D./105 cells, respectively; n = 4). Low PTP activity was observed in spermatozoa incubated in the presence of NEM (0.20 ± 0.06 O.D./105 cells; n = 4). If the activity in the presence of NEM is ascribed to PTP-independent hydrolysis of p-NPP, then the net PTP activity in the caput was about three-fold higher than in the cauda (0.75 ± 0.03, and 0.23 ± 0.13 O.D./105 cells, respectively; P < 0.01).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the rat, caput spermatozoa are immature and exhibit high thiol levels, whereas in the cauda, where spermatozoa acquire fertilizing ability, sperm thiols are oxidized. Moreover, oxidation of caput sperm protein thiols improved fertilizing ability, whereas reduction of cauda sperm thiols by DTT diminished fertilization [32, 38, 39].

It has been shown that tyrosine phosphorylation of spermatozoa from various species is increased following sperm incubation in certain culture medium [4, 23] and in capacitation medium [1, 22, 40, 41]. The enhanced tyrosine phosphorylation is associated with thiol oxidation of capacitated spermatozoa [11, 15].

In this paper we show that several sperm tail proteins of spermatozoa isolated from cauda epididymis exhibit higher tyrosine phosphorylation than proteins of spermatozoa isolated from caput epididymis. The fact that epididymal sperm tyrosine phosphorylation of some proteins can be enhanced by thiol oxidation and diminished by reducing disulphides supports the notion that thiol status plays a role in tyrosine phosphorylation during epididymal sperm maturation, as it does later during sperm capacitation. Our results support previous immunohistochemical results that have demonstrated that caput spermatozoa show only occasional labeling of tyrosine phosphorylation on the sperm tail, whereas cauda spermatozoa show significant tail phosphotyrosine labeling [4].

Previous immunohistochemical analysis has demonstrated differences between caput and cauda in the localization of head tyrosine phosphorylated proteins and a decrease in sperm head phosphotyrosine residues [4, 23]. Our immunofluorescence results also show a change in the localization of head phosphoproteins, but associated with a moderate increase in phosphotyrosine level during maturation. The reasons for the quantitative differences observed are not clear at present. The overall results, however, show that during rat sperm epididymal maturation, phosphotyrosine proteins become more evident at the posterior margin of the sperm head. Although protamine contains tyrosine residues [42], tyrosine phosphorylation of this protein has not been studied. The results of the present work show tyrosine phosphorylation of protamine in caput and cauda sperm and indicate increased protamine phosphorylation during sperm maturation. The relation of the altered localization of phosphotyrosine proteins and protamine phosphorylation to chromatin condensation remains to be studied.

Protein tyrosine phosphorylation is controlled by the opposing activity of tyrosine kinases and phosphotyrosine phosphatases. It has been suggested that the regulation of tyrosine phosphorylation in mammalian spermatozoa is via cAMP/protein kinase A (PKA) pathway that appears to be unique to this cell type [1, 22, 23]. In the present work we demonstrated the contribution of phosphatase activity to sperm tyrosine phosphorylation during sperm maturation. Under native conditions, when PTK and PTP are active, low tyrosine phosphorylation is found in several caput sperm proteins as compared with cauda sperm. Following diminution in PTP activity, while kinases remain active, sperm tail protein tyrosine phosphorylation is increased in caput sperm. Our results are also consistent with the notion of a high PTP activity in caput sperm heads and diminution during passage of sperm from caput to cauda epididymis (along with a change from high to low sperm head thiol levels).

Only limited information about PTP in mammalian spermatozoa is available. Here, we demonstrate for the first time differences between caput and cauda spermatozoa in the PTP-1B protein profile. In the rat caput spermatozoa, we observed PTP protein bands of about 50, 40, and 27 kDa, whereas in the cauda spermatozoa mostly the 27 kDa band was observed. Because sperm proteins were analyzed under denaturation and reducing conditions (with SDS and DTT), it is unlikely that the observed loss of the PTP 50 kDa band in the cauda spermatozoa is due to the presence of high molecular mass, disulfide-containing proteins in the cauda spermatozoa. It is important to note that the differences between caput and cauda sperm PTP protein bands could not be reversed by reduction or oxidation of thiols in the isolated spermatozoa. PTP variants of various molecular masses have been found in different cell types [43–45]. Hormones, such as insulin, induce changes in the relative ratio of PTP1B splice variants [44]. Thus, hormones or other epididymal factors may affect posttranscription processing of PTP during sperm maturation and cause loss of the 50 kDa variant. Alternatively, PTP may undergo degradation in the maturing spermatozoa, as previously proposed for the presence of PTP variants in other cells [46]. Such degradation may be due to oxidation of PTP, as the oxidation of a variety of proteins is known to enhance their susceptibility to degradation [47] as well as to oxidation-induced enhanced protease activity [48]. The lower activity of PTP found in cauda spermatozoa as compared with PTP activity in caput spermatozoa is thus consistent with diminished activity of PTP due to oxidation and to PTP loss. Additional work is necessary to further study these aspects of epididymal sperm maturation.

Our results suggest that the 50 kDa PTP band is associated with sperm plasma membrane, whereas the lower molecular mass PTP bands are associated with internal sperm structures. Previous work has demonstrated a membrane-associated protein phosphatase in hamster spermatozoa [30]. The results presented here add to these findings and indicate that an association of PTP with sperm membranes is not unique to one species.

Overall, the differences in tyrosine phosphorylation between caput and cauda spermatozoa described here may in part be due to altered PTP. The relation of PTK and other factors to sperm tyrosine phosphorylation and sperm fertilizing ability remains to be studied.


   FOOTNOTES
 
1 Correspondence: Nechama S. Kosower, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel-Aviv University, Tel Aviv 69978, Israel. FAX: 972 3 640 9900; nkosower{at}post.tau.ac.il Back

Received: 31 January 2004.

First decision: 16 February 2004.

Accepted: 17 May 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Carrera A, Moos J, Ping Ning X, Gerton GL, Tesarik J, Kopf GS, Moss SB. Regulation of protein tyrosine phosphorylation in human sperm by calcium/calmodulin-dependent mechanism: identification of a kinase anchor protein as major substrates for tyrosine phosphorylation. Dev Biol 1996 180:284-296[CrossRef][Medline]
  2. Pukazhenthi BS, Long JA, Wildt DE, Ottinger MA, Armstrong DL, Howard J. Regulation of sperm function by protein tyrosine phosphorylation in diverse wild felid species. J Androl 1998 19:675-685[Abstract/Free Full Text]
  3. Si Y, Okuno M. Role of tyrosine phosphorylation of flagellar proteins in hamster sperm hyperactivation. Biol Reprod 1999 61:240-246[Abstract/Free Full Text]
  4. Baker M, Lewis B, Hetherington L, Aitken J. Development of the signaling pathways associated with sperm capacitation during epididymal maturation. Mol Reprod Dev 2003 64:446-457[CrossRef][Medline]
  5. Yanagimachi R. Mammalian fertilization. In: Knobil K, Neill JD (eds.), The Physiology of Reproduction. New York: Raven Press; 1994:189–317
  6. Tash JS. Protein phosphorylation: the second messenger signal transducer of flagellar motility. Cell Motil Cytoskel 1989 14:332-339[CrossRef][Medline]
  7. Tash JS, Bracho GE. Regulation of sperm motility: emerging evidence for a major role for protein phosphatases. J Androl 1994 15:505-509[Free Full Text]
  8. Brautigan DL. Great expectations: protein tyrosine phosphatases in cell regulation. Biochim Biophys Acta 1992 1114:63-77[Medline]
  9. Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 1995 80:225-236[CrossRef][Medline]
  10. Tonks NK. PTP1B: from the sidelines to the front lines!. FEBS Lett 2003 546:140-148[CrossRef][Medline]
  11. Aitken RJ, Paterson M, Fisher H, Buckingham DW, van Duin M. Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. J Cell Sci 1995 108:2017-2025[Abstract]
  12. Caselli A, Marzocchini R, Camici G, Manao G, Moneti G, Pieraccini G, Ramponi G. The inactivation mechanism of low molecular weight phosphotyrosine-protein phosphatase by H2O2. J Biol Chem 1998 273:32554-32560[Abstract/Free Full Text]
  13. Monteiro HP, Ivaschenko Y, Fischer R, Stern A. Inhibition of protein tyrosine phosphatase activity by diamide is reversed by epidermal growth factor in fibroblasts. FEBS Lett 1991 295:146-148[CrossRef][Medline]
  14. Zipser Y, Piade A, Kosower NS. Erythrocyte thiol status regulates band 3 phosphotyrosine level via oxidation/reduction of band 3-associated phosphotyrosine phosphatase. FEBS Lett 1997 406:126-130[CrossRef][Medline]
  15. De Lamirande E, Gagnon C. Paradoxical effect of reagents for sulfhydryl and disulfide groups on human sperm capacitation and superoxide production. Free Rad Biol Med 1998 25:803-817[CrossRef][Medline]
  16. Peranovich TM, da Silva AM, Fries DM, Stern A, Monteiro HP. Nitric oxide stimulates tyrosine phosphorylation in murine fibroblasts in the absence and presence of epidermal growth factor. Biochem J 1995 305:613-619
  17. Thundathil J, de Lamiranda E, Gagnon C. Nitric oxide regulates the phosphorylation of the threonine-glutamine-tyrosine motif in proteins of human spermatozoa during capacitation. Biol Reprod 2003 68:1291-1298[Abstract/Free Full Text]
  18. Gross S, Knebel A, Tenev T, Neininger A, Gaestel M, Herrlich P, Bohmer FD. Inactivation of protein-tyrosine phosphatases as mechanism of UV-induced signal transduction. J Biol Chem 1999 274:26378-26386[Abstract/Free Full Text]
  19. Shalgi R, Seligman J, Kosower NS. Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescent labeling agent monobromobimane. Biol Reprod 1989 40:1037-1045[Abstract]
  20. Seligman J, Shalgi R. Protein thiols in spermatozoa and epididymal fluid of rats. J Reprod Fertil 1991 93:399-408
  21. Berruti G, Martegani E. Identification of proteins cross-reactive to phosphotyrosine antibodies and of tyrosine kinase activity in boar spermatozoa. J Cell Sci 1989 93:667-674[Abstract/Free Full Text]
  22. Visconti PE, Bailey JL, Moore GD, Pan D, Old-Clarke P, Kopf GS. Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development 1995 121:1129-1137[Abstract]
  23. Lewis B, Aitken RJ. Impact of epididymal maturation on the tyrosine phosphorylation patterns exhibited by rat spermatozoa. Biol Reprod 2001 64:1545-1556[Abstract/Free Full Text]
  24. Gamou S, Shimizu N. Hydrogen peroxide preferentially enhances the tyrosine phosphorylation of epidermal growth factor receptor. FEBS Lett 1995 357:161-164[CrossRef][Medline]
  25. Guyton KZ, Liu Y, Gorospe M, Xu Q, Holbrook NJ. Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury. J Biol Chem 1996 271:4138-4142[Abstract/Free Full Text]
  26. Van Montfort RL, Congreve M, Tisi D, Carr R, Jhoti H. Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B. Nature 2003 423:773-777[CrossRef][Medline]
  27. Heffetz D, Bushkin I, Dror R, Zick Y. The insulinomimetic agents H2O2 and vanadate stimulate protein tyrosine phosphorylation in intact cells. J Biol Chem 1990 265:2896-2902[Abstract/Free Full Text]
  28. Devi KU, Ahmad MB, Shivaji S. A maturation-related differential phosphorylation of the plasma membrane proteins of the epididymal spermatozoa of the hamster by endogenous protein kinases. Mol Reprod Dev 1997 47:341-350[CrossRef][Medline]
  29. Huacuja L, Delgado NM, Merchant H, Pancardo RM, Rosado A. Cyclic AMP induced incorporation of 3 3Pi into human spermatozoa membrane components. Biol Reprod 1977 17:89-96[Abstract]
  30. Devi KU, Jha K, Shivaji S. Plasma membrane-associated protein tyrosine phosphatase activity in hamster spermatozoa. Mol Reprod Dev 1999 53:42-50[CrossRef][Medline]
  31. Zipser Y, Kosower NS. Phosphotyrosine phosphatase associated with band 3 protein in the human erythrocyte membrane. Biochem J 1996 314:881-887
  32. Seligman J, Kosower NS, Shalgi R. Effects of caput ligation on rat sperm and epididymis: protein thiols and fertilizing ability. Biol Reprod 1992 46:301-308[Abstract]
  33. Panyim S, Chalkley R. High resolution acrylamide gel electrophoresis of histones. Arch Biochem Biophys 1969 130:337-346[CrossRef][Medline]
  34. Clari G, Brunati AM, Moret V. Membrane-bound phosphotyrosyl-protein phosphatase activity in human erythrocytes. Dephosphorylation of membrane band 3 protein. Biochem Biophys Res Comm 1987 142:587-594[CrossRef][Medline]
  35. Aviles M, Abascal I, Martinez-Menarguez JA, Castells MT, Skalaban SR, Ballesta J, Alhadeff JA. Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum alpha-L-fucosidase from rat testis and epididymal spermatozoa. Biochem J 1996 318:821-831
  36. Bennett V, Stenbuck PJ. The membrane attachment protein for spectrin is associated with band 3 in human erythrocyte membranes. Nature 1979 280:468-473[CrossRef][Medline]
  37. Kaneko T, Whittingham DG, Overstreet JW, Yanagimachi R. Tolerance of the mouse sperm nuclei to freeze-drying depends on their disulfide status. Biol Reprod 2003 69:1859-1862[Abstract/Free Full Text]
  38. Seligman J, Kosower NS, Shalgi R. Effects of castration on thiol status in rat spermatozoa and epididymal fluid. Mol Reprod Dev 1997 47:295-301[CrossRef][Medline]
  39. Rho GJ, Kawarsky S, Johnson WH, Kochhar K, Betteridge KJ. Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes. Biol Reprod 1998 59:918-924[Abstract/Free Full Text]
  40. Ficarro S, Chertihin O, Westbrook VA, White F, Jayes F, Kalab P, Marto JA, Shabanowitz J, Herr JC, Hunt DF, Visconti PE. Phosphoproteome analysis of capacitated human sperm. J Biol Chem 2003 278:11579-11589[Abstract/Free Full Text]
  41. Ecroyd HW, Jones RC, Aitken RJ. Endogenous redox activity in mouse spermatozoa and its role in regulating the tyrosine phosphorylation events associated with sperm capacitation. Biol Reprod 2003 69:347-354[Abstract/Free Full Text]
  42. Tanhauser SM, Hecht NB. Nucleotide sequence of the rat protamine 2 gene. Nucleic Acids Res 1989 17:4395[Free Full Text]
  43. Modesti A, Marzocchini R, Raugei G, Chiti F, Sereni A, Magherini F, Ramponi G. Cloning, expression and characterisation of a new human low Mr phosphotyrosine protein phosphatase originating by alternative splicing. FEBS Lett 1998 431:111-115[CrossRef][Medline]
  44. Sell SM, Reese D. Insulin-inducible changes in the relative ratio of PTP1B splice variants. Mol Genet Metab 1999 66:189-192[CrossRef][Medline]
  45. Lucentini L, Fulle S, Ricciolini C, Lancioni H, Panara F. Low molecular weight phosphotyrosine protein phosphatase from PC12 cells. Purification, some properties and expression during neurogenesis in vitro and in vivo. Int J Biochem Cell Biol 2003 35:1378-1387[CrossRef][Medline]
  46. Colas B, Cambillau C, Buscail L, Zeggari M, Esteve JP, Lautre V, Thomas F, Vaysse N, Susini C. Stimulation of a membrane tyrosine phosphatase activity by somatostatin analogues in rat pancreatic acinar cells. Eur J Biochem 1992 207:1017-1024[Medline]
  47. Moskovitz J, Yim MB, Chock PB. Free radicals and disease. Arch Biochem Biophys 2002 397:354-359[CrossRef][Medline]
  48. Cai J, Jones DP. Mitochondrial redox signaling during apoptosis. J Bioenerg Biomembr 1999 31:327-334[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
R. Jones, P. S. James, D. Oxley, J. Coadwell, F. Suzuki-Toyota, and E. A. Howes
The Equatorial Subsegment in Mammalian Spermatozoa Is Enriched in Tyrosine Phosphorylated Proteins
Biol Reprod, September 1, 2008; 79(3): 421 - 431.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
J. Martinez-Heredia, S. de Mateo, J. M. Vidal-Taboada, J. L. Ballesca, and R. Oliva
Identification of proteomic differences in asthenozoospermic sperm samples
Hum. Reprod., April 1, 2008; 23(4): 783 - 791.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
R. Oliva
Protamines and male infertility
Hum. Reprod. Update, July 1, 2006; 12(4): 417 - 435.
[Abstract] [Full Text] [PDF]

Home page
 J AndrolHome page
J. Seligman, G. L. Newton, R. C. Fahey, R. Shalgi, and N. S. Kosower
Nonprotein Thiols and Disulfides in Rat Epididymal Spermatozoa and Epididymal Fluid: Role of {gamma}-Glutamyl-Transpeptidase in Sperm Maturation
J Androl, September 1, 2005; 26(5): 629 - 637.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
K. Dvorakova, H. D M Moore, N. Sebkova, and J. Palecek
Cytoskeleton localization in the sperm head prior to fertilization
Reproduction, July 1, 2005; 130(1): 61 - 69.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/3/1009    most recent
biolreprod.104.028035v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Seligman, J.
Right arrow Articles by Kosower, N. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Seligman, J.
Right arrow Articles by Kosower, N. S.
Agricola
Right arrow Articles by Seligman, J.
Right arrow Articles by Kosower, N. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS